Entamoeba histolytica, a protozoan parasite, is an important human pathogen. Diseases caused by E. histolytica include dysentery and liver abscesses, and the organism is a leading parasitic cause of death on a global scale. Regulation of gene expression is a key factor that enables the parasite to adapt to the host environment during tissue invasion and to convert to the cyst stage and propagate disease outside the host. One mechanism of gene regulation in Entamoeba is a robust and complex endogenous RNAi pathway. Over the last several years we have made important observations about this pathway including that small RNAs associate with amebic Argonaute protein to mediate transcriptional gene silencing and have highly unusual 5'- polyP termini. However, despite extensive efforts we have not identified which aspects of amebic biology are regulated by small RNAs, although given that the pathway is maintained in related Entamoeba, a strong evolutionary pressure and clear biological role must exist. We have recently identified that ameba secrete extracellular vesicles that contain small RNAs and RNAi effector proteins. We now seek to determine whether these extracellular vesicles containing small RNAs serve as a means of host-parasite or parasite-parasite intercellular communication. Thus, we will (i) identify small RNA repertoire of amebic extracellular vesicles, (ii) determine if they participate in intercellular communication, and (iii) determine protein components of EVs with focus on RNA binding proteins. These data will improve our understanding of the molecular mechanisms of RNAi in Entamoeba and will also broaden our understanding of the roles of small RNAs in intercellular communication. Our work is at the intersection of the basic cellular process of RNA- interference and amebic biology. Data that emerge will contribute to both understanding amebic pathogenesis but also to expanding the knowledge about the fundamental process of RNAi.